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Detailed glycan structural characterization is frequently achieved by collisionally activated dissociation (CAD) based sequential tandem mass spectrometry (MS n ) analysis of permethylated glycans. However, it is challenging to implement MS n ( n &gt; 2) during online glycan separation, and this has limited its application to analysis of complex glycan mixtures from biological samples. Further, permethylation can reduce liquid chromatographic (LC) resolution of isomeric glycans. Here, we studied the electronic excitation dissociation (EED) fragmentation behavior of native glycans with a reducing-end fixed charge tag and identified key spectral features that are useful for topology and linkage determination. We also developed a de novo glycan sequencing software that showed remarkable accuracy in glycan topology elucidation based on the EED spectra of fixed charge-derivatized glycans. The ability to obtain glycan structural details at the MS 2 level, without permethylation, via a combination of fixed charge derivatization, EED, and de novo spectral interpretation, makes the present approach a promising tool for comprehensive and rapid characterization of glycan mixtures.

De Novo Glycan Sequencing by Electronic Excitation Dissociation and Fixed-Charge Derivatization